The liver faces a major challenge of free fatty acid (FFA) influx. To prevent the potential hepatic lipotoxicity, the liver efficiently converts FFA to triacylglycerol (TAG) for storage, oxidizes them to ketones, or exports them as very-low density lipoproteins (VLDLs). TAG utilized to assemble VLDL is crucial for the distribution of lipids to peripheral tissues for energy in vertebrates. On metabolism of VLDL, however, LDL is generated which is the major carrier of cholesterol and is associated with the development of atherosclerosis. The movement of VLDL from its site of synthesis, the endoplasmic reticulum (ER) to the Golgi is required for its eventual secretion from the hepatocyte and represents the potential therapeutic target in controlling elevated concentrations of LDL in the plasma, the determinant of atherosclerosis. To understand how VLDL exits from the ER and its delivery to the Golgi at the molecular level is the subject of the current application. Our preliminary studies show that VLDL and albumin (a typical liver secretory protein) are transported separately in two different vesicles from the liver ER to the Golgi. Despite the two cargoes being transported in different vesicles, the same initiator of ER vesicle budding, Sar1, is utilized by both types of vesicles. This suggests that a specialized vesicle is utilized for VLDL and that different proteins are involved with the selection of cargo for each of the two types of transport vesicles. The first aim of this grant is to identify the cargo selecting protein(s) required for the inclusion of VLDL in the VLDL-transport-vesicle (VTV). 2D-DIGE analyses revealed that VTV contains 5 proteins that are not present in protein vesicles. In preliminary studies we have identified Sar1b as the initiator of VTV budding. Further, our data show apolipoprotein B (apoB) to be the cargo selective protein on ER-VLDL. Our second aim will be to identify the pre-budding complex that forms to bud VTV from the liver ER. Preliminary studies show that VTVs greatly differ from albumin containing vesicles in their size, density and protein composition. Because of the major differences between the two carrier vesicles, the VTV may have different proteins on its surface, which are required for docking and fusion of the VTV with the cis Golgi. We propose to identify the SNARE proteins responsible for the targeting/docking of VTVs to the liver Golgi and their cognate SNARE proteins on the liver Golgi. Our preliminary data show that fusion of VTVs with the liver Golgi requires cytosolic factor(s). Further, VTVs do not fuse with the liver Golgi when cytosol treated with proteinase K or boiled cytosol was used, suggesting that the cytosolic factor(s) required for fusion to occur is a protein(s). Another aim of this proposal is to determine cytosolic proteins that control the delivery of VLDL to the Golgi. Project Narrative: Transport of VLDL from its site of synthesis, the endoplasmic reticulum to the Golgi is required in its eventual secretion from the hepatocyte and represents a potential therapeutic target in controlling elevated concentrations of LDL, the major carrier of cholesterol and determinant of atherosclerosis. The identification of proteins involved in the process of VLDL selection into VTVs, VTV budding and its fusion with Golgi would offer potential targets for their inhibition and thus potentially to control VLDL secretion from the liver.